U.S. patent number 7,858,337 [Application Number 12/074,329] was granted by the patent office on 2010-12-28 for process for the manufacture of a composite material.
This patent grant is currently assigned to Novartis AG. Invention is credited to Hans-Lothar Fuchsbauer, Monika Knuth, Achim Muller, Kai Oertel, Ralf Pasternack, Christine Reiff, geb. Schmitt, Katharina Schmid, Jens Zotzel.
United States Patent |
7,858,337 |
Muller , et al. |
December 28, 2010 |
Process for the manufacture of a composite material
Abstract
The invention relates to a process for the manufacture of a
composite materials comprising the steps of (a) providing a
hydrophobic organic bulk material, and (b) applying a hydrophilic
surface coating on the hydrophobic organic bulk material by first
non-covalently attaching to the surface of the bulk material a
water-soluble peptide comprising a hydrophobic moiety; and then
chemically or enzymatically crosslinking the water-soluble peptide.
The composite materials manufactured according to the process of
the invention have desirable characteristics regarding adherence to
the substrate, durability, hydrophilicity, wettability,
biocompatibility and permeability and are thus particularly useful
as ophthalmic devices.
Inventors: |
Muller; Achim (Grossostheim,
DE), Knuth; Monika (Aschaffenburg, DE),
Schmid; Katharina (Aschaffenburg, DE), Pasternack;
Ralf (Griesheim, DE), Zotzel; Jens (Darmstadt,
DE), Oertel; Kai (Mainz, DE), Reiff, geb.
Schmitt; Christine (Lorrach, DE), Fuchsbauer;
Hans-Lothar (Muhltal, DE) |
Assignee: |
Novartis AG (Basel,
CH)
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Family
ID: |
38128157 |
Appl.
No.: |
12/074,329 |
Filed: |
March 3, 2008 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090029413 A1 |
Jan 29, 2009 |
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Foreign Application Priority Data
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Mar 8, 2007 [EP] |
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07103810 |
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Current U.S.
Class: |
435/68.1;
530/300; 435/41; 530/329 |
Current CPC
Class: |
C08J
7/043 (20200101); G02C 7/04 (20130101); A61L
27/50 (20130101); A61L 27/28 (20130101); A61L
27/34 (20130101); C08J 7/046 (20200101); C08J
7/0427 (20200101); C08J 7/056 (20200101); A61L
27/34 (20130101); C08L 89/00 (20130101); C08J
2489/00 (20130101); A61F 2/14 (20130101) |
Current International
Class: |
C07K
7/00 (20060101); C12P 21/04 (20060101); C12P
1/00 (20060101) |
Field of
Search: |
;435/68.1,41
;530/300,329 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 00/18794 |
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Apr 2000 |
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WO |
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WO 01/56627 |
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Aug 2001 |
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WO |
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WO 2004/050132 |
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Jun 2004 |
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WO |
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Other References
PCT International Search Report, (Sep. 2008). cited by other .
PCT Written Opinion of the International Searching Authority, (Sep.
2008). cited by other .
European Search Report, (Sep. 2007). cited by other .
Examiners Communication. cited by other.
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Primary Examiner: Kam; Chih-Min
Attorney, Agent or Firm: Zhou; Jian
Claims
The invention claimed is:
1. A process for the manufacture of a composite material,
comprising the steps of: (a) providing a hydrophobic organic bulk
material; (b) applying a hydrophilic surface coating on the
hydrophobic organic bulk material by first non-covalently attaching
to the surface of the bulk material a water-soluble peptide
comprising a hydrophobic moiety, wherein the water-soluble peptide
is of the formula A-C(O)-[NH-(peptide)-C(O)]-X.sub.1-R.sub.3 (3),
wherein A-C(O) is a radical selected from the group consisting of
the radical of a fatty acid, the radical of an aromatic carboxylic
acid and the radical of an araliphatic carboxylic acid,
[NH-(peptide)-C(O)] is the radical of a polypeptide which is a
statistical copolymer of lysine and more than one other amino acids
selected from the group consisting of alanine, phenylalanine,
serine, tyrosine and tryptophan, and wherein the molecular weight
range M.sub.r of the polypeptide is from 400 to 10000 daltons,
X.sub.1--R.sub.3 is either OH and part of the terminal peptide
carboxy group, or X.sub.1 is O or NR.sub.4 wherein R.sub.4 is
hydrogen or C.sub.1--C.sub.2-alkyl, and R.sub.3 is a hydrophilic
group; and (c) then chemically or enzymatically crosslinking said
water-soluble peptide.
2. The process according to claim 1, wherein the hydrophobic
organic bulk material is a polysiloxane, perfluoroalkyl polyether,
fluorinated poly(meth)acrylate, polyalkyl (meth)acrylate,
fluorinated polyolefin or a mixture thereof.
3. The process according to claim 1, wherein the hydrophobic
organic bulk material is a polysiloxane hydrogel, a perfluoroalkyl
polyether hydrogel or a mixture thereof.
4. The process according to claim 1, wherein the polypeptide
underlying the polypeptide radical [NH-(peptide)-C(O)] is the
radical of a polypeptide having from 3 to 20 amino acid residues
and having a statistical composition consisting of one tyrosine
residue, 1 to 8 alanine residues, and 1 to 20 lysine residues.
5. The process according to claim 4, wherein the polypeptide
underlying the polypeptide radical [NH-(peptide)-C(O)] is a
statistical copolymer consisting of one tyrosine (Tyr) residue, 3
alanine (Ala) residue, and 3 lysine (Lys) residues.
6. The process according to claim 4, wherein the hydrophilic
surface coating is finalized by chemically initiating the
crosslinking of the water soluble peptide being attached to the
organic bulk material with a crosslinking agent selected from
formaldehyde or glutaraldehyde.
7. The process according to claim 4, wherein the hydrophilic
surface coating is finalized by enzymatic crosslinking of the water
soluble peptide being attached to the organic bulk material with a
peptide substrate of transglutaminase.
8. The process according to claim 1, wherein the polypeptide
underlying the polypeptide radical [NH-(peptide)-C(O)] is a
copolymer of the SEQ ID NO 1: Tyr-Ala-Lys-Ala-Lys-Lys-Ala (4c),
wherein Tyr is linked to A, and Ala is linked to R.sub.3.
9. The process according to claim 1, wherein the hydrophilic
surface coating is finalized by chemically initiating the
crosslinking of the water soluble peptide being attached to the
organic bulk material with a crosslinking agent selected from
formaldehyde or glutaraldehyde.
10. The process according to claim 1, wherein the hydrophilic
surface coating is finalized by enzymatic crosslinking of the water
soluble peptide being attached to the organic bulk material with a
peptide substrate of transglutaminase.
11. The process according to claim 10, wherein the peptide
substrate is a peptide that contains glutamine.
12. The process according to claim 11, wherein the composite
material is a contact lens.
13. The process according to claim 12, wherein the polypeptide
underlying the polypeptide radical [NH-(peptide)-C(O)] is a
copolymer of the SEQ ID NO 1: Tyr-Ala-Lys-Ala-Lys-Lys-Ala (4c),
wherein Tyr is linked to A, and Ala is linked to R.sub.3.
14. The process according to claim 1, wherein the polypeptide
underlying the polypeptide radical [NH-(peptide)-C(O)] is a
statistical copolymer of lysine and an amino acid selected from the
group consisting of alanine, and tyrosine.
Description
This application claims benefit under 35 USC .sctn.119 of European
patent application No. EP 07103810.3 filed Mar. 8, 2007, the
contents of which are incorporated herein by reference in its
entirety.
INCORPORATION OF SEQUENCE LISTING
A paper copy of the Sequence Listing and a copy of the Sequence
Listing on diskette, containing the file named
50700_US_NP_ST25.txt, which is 460 bytes in size (measured in
MS-DOS) and created on Aug. 20, 2008, is herein incorporated by
reference.
The present invention relates to a process for the manufacture of
coated articles such as biomedical articles, especially contact
lenses, which comprises at least partly coating said article with a
crosslinkable hydrophilic peptide, and then chemically or
enzymatically crosslinking said hydrophilic peptide.
A variety of different types of processes for preparing coatings on
an "inert" hydrophobic substrate have been disclosed in the prior
art. For example, WO-A-2004/050132 discloses to first of all
provide a hydrophobic uncharged article surface with some bilayers
composed of a polyacrylic acid and a polyallylamine hydrochloride
and then to covalently attach an antibacterial peptide to the
acidic component of the bilayers. However, the formation of the
bilayers is time-consuming and their stability, in particular their
long-term stability, is sometimes not totally satisfactory. This
may in turn affect the wearer comfort of a biomedical article when
worn in or on the human body, for example on the eye.
US-A-2006/0134166 discloses a method for making a non-crosslinked
biodegradable copolymer coating on a surface of a medical device,
wherein the copolymer is a polyamino acid which is derivatized to
have a hydrophobic side chain.
US-A-2004/0224080 generally discloses an enzymatically crosslinked
surface coating on a medical device, wherein lysine and glutamine
are enzymatically crosslinked by use of transglutaminase.
US-A-2003/0175745 discloses that polypeptides can be used to coat
solid surfaces of a biomedical device. It further discloses that
peptides can be chemically crosslinked with glutaraldehyde.
In addition, known coating processes are in general batch
processes, which are expensive to perform and which require
extensive handling steps. Because of this, none of the existing
processes is, for example, well suited for the integration into a
fully automated high volume contact lens manufacturing process as
described, for example, in EP-A-969956 or EP-A-1047542.
Accordingly, there is a need to provide new hydrophilic coatings on
a hydrophobic biomedical article surface which on the one hand have
an improved durability and cause an improved wearer comfort of the
biomedical article, and which on the other hand may be manufactured
in an easy way so as to be integrable in a mass manufacturing
process.
Surprisingly, it has now been found, that hydrophobic articles may
be rendered effectively hydrophilic on their surface by first
non-covalently binding a peptide to the article surface and then
subjecting said peptide to a crosslinking reaction.
The present invention therefore in one aspect relates to a process
for the manufacture of a composite material comprising the steps of
(a) providing a hydrophobic organic bulk material; and (b) applying
a hydrophilic surface coating on said bulk material by first
non-covalently attaching to the surface of the bulk material a
water-soluble peptide comprising a hydrophobic moiety; and then
chemically or enzymatically crosslinking said water-soluble
peptide.
The hydrophobic organic bulk material underlying the composite
materials is preferably a material that is devoid of ionic groups
such as cationic or anionic groups or has at least a relatively low
concentration of ionic groups. Accordingly, the surface of the
preferred bulk materials also has a low concentration of ionic
groups or is even devoid of ionic groups such as carboxy, sulfo,
amino and the like groups and thus may be substantially free of
ionic charges.
Examples of suitable bulk materials are natural or synthetic
organic polymers or modified biopolymers which are known in large
number. Some examples of polymers are polyaddition and
polycondensation polymers (polyurethanes, epoxy resins, polyethers,
polyesters, polyamides and polyimides); vinyl polymers
(polyacrylates, polymethacrylates, polyacrylamides,
polymethacrylamides, polystyrene, polyethylene and halogenated
derivatives thereof, polyvinyl acetate and polyacrylonitrile); or
elastomers (silicones, polybutadiene and polyisoprene).
A preferred group of materials to be coated are those being
conventionally used for the manufacture of biomedical devices, e.g.
contact lenses, in particular contact lenses for extended wear,
which are not hydrophilic per se. Such materials are known to the
skilled artisan and may comprise for example polysiloxanes,
perfluoroalkyl polyethers, fluorinated poly(meth)acrylates,
polyalkyl (meth)acrylates, or fluorinated polyolefines, such as
fluorinated ethylene or propylene, for example tetrafluoroethylene,
preferably in combination with specific dioxols, such as
perfluoro-2,2-dimethyl-1,3-dioxol. Mixtures of two or more of the
above-mentioned materials are also possible.
Within the present invention polysiloxane hydrogels, perfluoroalkyl
polyether hydrogels or mixtures thereof, in particular polysiloxane
hydrogels, are the preferred hydrophobic organic bulk
materials.
Examples of suitable polysiloxane hydrogels are, for example, those
currently used for the manufacture of extended wear contact lenses,
for example copolymers of (i) one or more hydrophilic monomers, for
example selected from the group of hydroxyethylacrylate,
hydroxyethylmethacrylate, acrylamide, N,N-dimethyl acrylamide,
N-vinylpyrrolidone, acrylic or methacrylic acid, and (ii) a
siloxane monomer and/or macromonomer, for example
tris-trimethylsilyloxy-silyl-propyl methacrylate (TRIS), or a
polysiloxane crosslinker, for example, as described in formula (2)
below. Examples of suitable commercially available silicon
hydrogels are Balafilcon A, Galyfilcon A, Lotrafilcon A,
Lotrafilcon B or Senofilcon A.
Another group of preferred polysiloxane hydrogels are amphiphilic
segmented copolymers comprising at least one hydrophobic siloxane
or perfluoroalkyl polyether segment and at least one hydrophilic
segment which are linked through a bond or a bridge member.
Examples of said polysiloxane hydrogels are disclosed, for example,
in PCT applications WO-A-96/31792 and WO-A-97/49740. A particularly
preferred amphiphilic segmented copolymer comprises at least one
hydrophobic segment selected from the group consisting of a
polysiloxane, perfluoroalkyl polyether and a mixed
polysiloxane/perfluoroalkyl polyether segment, and at least one
hydrophilic segment selected from the group consisting of a
polyoxazoline, poly(2-hydroxyethylacrylate),
poly(2-hydroxyethylmethacrylate), polyacrylamide,
poly(N,N-dimethylacrylamide), polyvinylpyrrolidone and a
polyethyleneoxide segment.
Still another group of preferred polysiloxane hydrogels are those
obtainable by crosslinking a crosslinkable or polymerizable
prepolymer that is obtainable by (a) copolymerizing at least one
hydrophilic monomer having one ethylenically unsaturated double
bond and at least one siloxane crosslinker comprising two or more
ethylenically unsaturated double bonds in the presence of a chain
transfer agent having a functional group; and (b) reacting one or
more functional groups of the resulting copolymer with an organic
compound having an ethylenically unsaturated group. Polysiloxane
hydrogels of this type are disclosed, for example in
WO-A-01/71392.
A particularly preferred polysiloxane hydrogel is obtained by
crosslinking a prepolymer which is obtainable by (a) copolymerizing
a hydrophilic monomer of the formula
##STR00001## wherein R.sub.1 is hydrogen or methyl, and R.sub.2 is
--COO--(CH.sub.2).sub.2--OH, --CONH.sub.2, --CON(CH.sub.3).sub.2,
or
##STR00002## optionally in admixture with one or more further
hydrophilic monomers; and a polysiloxane crosslinker corresponds to
formula
##STR00003## wherein d.sub.1 is an integer from 10 to 500,
preferably 10 to 300, more preferably 20 to 200 and in particular
25 to 150, (alk) is linear or branched C.sub.2-C.sub.4 alkylene or
a radical --(CH.sub.2).sub.1-3--O--(CH.sub.2).sub.1-3--, X is --O--
or --NH-- and Q is a radical of the formula
##STR00004## in the presence of a chain transfer agent having a
functional group, in particular 2-mercaptoethanol or especially
2-aminoethane thiol (cysteamine); and (b) reacting the resulting
copolymer with an organic compound having an ethylenically
unsaturated group, for example with 2-isocyanatoethylmethacrylate
(IEM), 2-vinyl-azlactone, 2-vinyl-4,4-dimethyl-azlactone, acryloyl
or methacryloyl chloride, 2-hydroxyethylacrylate (HEA),
2-hydroxymethacrylate (HEMA), glycidylacrylate or
glycidylmethacrylat, in particular with IEM or acryloyl
chloride.
The water-soluble peptide being attached to the bulk material
surface is, for example, a peptide of the formula
A-C(O)--[NH-(peptide)-C(O)]--X.sub.1--R.sub.3 (3), wherein A-C(O)
is a hydrophobic radical, for example a radical selected from the
group consisting of the radical of a fatty acid, the radical of an
aromatic carboxylic acid and the radical of an araliphatic
carboxylic acid, [NH-(peptide)-C(O)] is the radical of a
polypeptide having an amino acid sequence comprising three or more
amino acids, at least one of them being lysine (Lys) or glutamine
(Gln), X.sub.1-R.sub.3 is either OH and part of the terminal
peptide carboxy group, or X.sub.1 is O or NR.sub.4 wherein R.sub.4
is hydrogen or C.sub.1-C.sub.2-alkyl, and R.sub.3 is a hydrophilic
group.
A-C(O) as the radical of a fatty acid is, for example, the radical
of a long-chain aliphatic monocarboxylic acid, which contains, for
example, from 6 to 25 carbon atoms and optionally comprises one or
more carbon-carbon double bonds. Preferably, A-C(O) is the radical
of a monocarboxylic acid of the formula C.sub.nH.sub.2n+1COOH,
wherein n is a number from 7 to 20 and in particular from 8 to 18.
Examples of preferred fatty acid radicals A-C(O) are the acyl
radical of caprinic acid, laurinic acid, palmitinic acid or
stearinic acid.
Examples of suitable aromatic acid radicals A-C(O) are the acyl
radical of an optionally substituted benzoic acid or of a naphthoic
acid.
Examples of a suitable radical of an araliphatic acid A-C(O) are
the radical of an optionally substituted phenyl acetic or propionic
acid or the radical of an 1- or 2-naphthyl acetic or propionic
acid.
Optional substitutents of the benzoic, phenylacetic or
phenylpropionic acid are, for example, C.sub.1-C.sub.2-alkyl or
C.sub.1-C.sub.2-alkoxy.
The amino acid sequence (peptide) in general may be the radical of
any water-soluble peptide which is chemically or enzymatically
crosslinkable.
The amino acid sequence underlying the polypeptide radical
[NH-(peptide)-C(O)] is preferably a sequence which may function as
a substrate for the enzyme transglutaminase, that is it contains
lysine and/or glutamine in form of a transglutaminase leader
sequence which may be crosslinked enzymatically.
Examples of suitable peptides underlying the polypeptide radical
[NH-(peptide)-C(O)] are a protein hydrolysate, for example a casein
hydrolysate; a glutene peptide; a polylysine; or a copolymer of
lysine and one or more other amino acids, for example, selected
from the group consisting of alanine, phenylalanine, serin,
tyrosine and tryptophane.
The molecular weight of the polypeptide underlying the radical
[NH-(peptide)-C(O)] is in general not critical but preferably has a
value M.sub.r of .ltoreq.12000. A preferred molecular weight range
of M.sub.r is from 400 to 10000, more preferably from 400 to 5000
and in particular from 500 to 1500.
One group of preferred polypeptides underlying the radical
[NH-(peptide)-C(O)] are polylysines; the molecular weight of said
polylysines is preferably within the above-given ranges including
the preferences.
A further group of preferred polypeptides are statistical
copolymers of lysine and one or more amino acids selected from the
group consisting of alanine, phenylalanine, serin, tyrosine and
tryptophane, wherein again the above given ranges and preferences
for the molecular weight apply.
A further suitable lysine copolymer has a statistical composition
consisting of: from zero to one, preferably one tyrosine (Tyr)
residue; from 0 to 8, preferably from 1 to 5 and in particular from
2 to 4 alanine (Ala) residues; from 1 to 20, preferably from 2 to
10, and in particular from 2 to 5 lysine (Lys) residues; the total
number of the amino acid residues in the lysine copolymer is from 3
to 20, preferably from 4 to 12 and in particular from 5 to 8.
Within this group of lysine copolymers, [NH-(peptide)-C(O)] is
preferably the radical of a polypeptide having a statistical
composition consisting of one tyrosine (Tyr) residue, 3 alanine
(Ala) residues, and 3 lysine (Lys) residues and even more preferred
the radical of a synthetic polypeptide of the SEQ ID NO 1:
Tyr-Ala-Lys-Ala-Lys-Lys-Ala wherein Tyr is linked to A, and Ala is
linked to R.sub.3.
A further group of preferred polypeptides underlying the radical
[NH-(peptide)-C(O)] comprises a glutene peptide comprising
glutamine units.
Still a further group of preferred polypeptides underlying the
radical [NH-(peptide)-C(O)] comprises a casein hydrolysate
comprising glutamine and lysine units.
X.sub.1 in formula (3) is preferably O or NH, in particular NH.
R.sub.3 as a hydrophilic group denotes, for example, hydrogen or a
C.sub.1-C.sub.6-alkyl radical which is substituted once or several
times by sulfo, sulfato, phosphato and/or carboxy. The terms sulfo,
sulfato, phosphato and carboxy in general include the free acid as
well as biomedically acceptable, in particular opthalmically
acceptable, salts thereof, for example sodium, potassium, magnesium
or ammonium salts. Preferably, R.sub.3 as a hydrophilic group
denotes a C.sub.1-C.sub.4-alkyl radical which is mono- or
disubstituted by sulfo, sulfato and/or carboxy. More preferably,
R.sub.3 as a hydrophilic group is a C.sub.2-C.sub.3-alkyl radical
which is monosubstituted by sulfo or carboxy. Most preferably,
R.sub.3 as hydrophilic group is 2-sulfoethyl.
In case X.sub.1-R.sub.3 is OH, formula (3) is meant to cover as
well suitable salts of the terminal carboxy group, for example the
sodium, potassium or an ammonium salt.
According to a preferred embodiment of the invention there is
attached to the hydrophobic organic bulk material in step (b) a
water-soluble peptide of the above-given formula (3), wherein
A-C(O) is the radical of a monocarboxylic acid of the formula
C.sub.nH.sub.2n+1COOH, wherein n is from 7 to 20;
[NH-(peptide)-C(O)] is an amino acid sequence derived from a
polypeptide having a molecular weight of from 400 to 10000 which is
selected from the group consisting of a protein hydrolysate, a
glutene peptide, a polylysine, or a copolymer of lysine and one or
more other amino acids, X.sub.1 is O or NH, and R.sub.3 is hydrogen
or C.sub.1-C.sub.4-alkyl which is mono- or disubstituted by sulfo,
sulfato and/or carboxy.
According to an even more preferred embodiment of the invention
there is attached to the hydrophobic organic bulk material in step
(b) a water-soluble peptide of the above-given formula (3), wherein
A-C(O) is the radical of a monocarboxylic acid of the formula
C.sub.nH.sub.2+1COOH, wherein n is from 8 to 18;
[NH-(peptide)-C(O)] is an amino acid sequence derived from a
polypeptide having a molecular weight of from 500 to 1500, which is
selected from the group consisting of a casein hydrolysate; a
glutene peptide; a polylysine; a statistical copolymer of lysine
and one or more amino acids selected from the group consisting of
alanine, phenylalanine, serin, tyrosine and tryptophane; and a
polypeptide of the formula Tyr-Ala-Lys-Ala-Lys-Lys-Ala (4c),
wherein Tyr is linked to A, and Ala is linked to R.sub.3, X.sub.1
is O or NH, and R.sub.3 is hydrogen or C.sub.2-C.sub.3-alkyl which
is monosubstituted by sulfo or carboxy.
The compounds of the formula (3) may be synthesized by methods
known per se. For example, a peptide of the formula
H.sub.2N-(peptide)-C(O)OH (3a), wherein (peptide) is as defined
above is reacted in any order with a compound of formula A-COOH
(5), wherein A is as defined before, and, if applicable, with a
compound of the formula R.sub.3--X.sub.1H (6), wherein R.sub.3 and
X.sub.1 are as defined above.
The compounds of formulae (5) and (6) are known and in general
commercially available. The peptides of formula (3a) can be
obtained in part from commercial suppliers or can be synthesized
according to any known suitable method. For example polylysines and
statistical copolymers of lysine and another amino acid may be
obtained by standard copolymerization reaction. Specific polylysine
copolymers may be obtained by solid phase peptide synthesis as
described, for example, in W. C. Chan and P. D. White, Fmoc Solid
Phase Peptide Synthesis, Practical Approach Series, Oxford
University Press. For example, the peptides underlying the
sequences of formulae (4a), (4b) and (4c), i.e. SEQ ID NO 1, can be
synthesized by reacting the underlying protected amino
acids--protected, for example, with the fluorenylmethoxycarbonyl
(Fmoc) radical--one after another immobilized at a polymeric
carrier, for example a polystyrene resin comprising chlorotrityl
anchors. Accordingly, a first protected amino acid, for example
Fmoc- and Boc (tert.-butylcarbonyl)-protected lysine or
Fmoc-protected alanine, is coupled to the polymeric carrier. After
completion of the coupling--which may be checked with the Kaiser
test--said amino acid is deprotected--typically with diluted
trifluoroacetic acid or with piperidine--before the coupling of the
second protected amino acid is initiated. Following the coupling
and deprotection of the last amino acid, the resulting peptide is
separated from the polymeric carrier in a manner known per se, for
example with a solution of trifluoroacetic acid in
dichloromethane.
The reactions of the compound of formula (3a) with the compounds of
the formula (5) and (6) are known per se from textbooks of Organic
Chemistry. In case the peptide of formula (3a) is prepared by solid
phase peptide synthesis as described above, the coupling of the
compound of the formula (5) to the peptide preferably can be added
to the peptide synthesis. Accordingly, to the peptide--before being
separated from the polymeric carrier--is coupled the compound of
formula (5) in the same manner as an amino acid.
The water-soluble peptide having the hydrophobic moiety is
non-covalently attached to the hydrophobic bulk material surface.
Accordingly attachment takes places, for example, by physical
absorption, physical incorporation into the polymer matrix of the
bulk material, complex formation, heteropolar bonding and/or by
ionic interactions.
The attachment of the peptide to the bulk material surface may be
accomplished according to processes known per se. For example, the
bulk material is immersed in a solution of the peptide, or one or
more layers of the peptide are deposited on the bulk material
surface, for example, by dipping, spraying, printing, spreading,
pouring, rolling or spin coating, spraying or particularly dipping
being preferred.
A suitable dip solution of the peptide in general comprises the
respective peptide diluted in one or more different solvents.
Suitable solvents are, for example, water or an aqueous solution
comprising a water-miscible organic solvent, for example THF or a
C.sub.1-C.sub.4-alkanol such as methanol, ethanol or isopropanol;
the preferred solvent is water. The pH of the aqueous solution of
the peptide is dependent of the specific polypeptide used. A
suitable buffer, for example a phosphate buffer, may be added to
the dip solution in order to maintain a constant pH value. The dip
solution may contain additional ingredients, for example salts. The
concentration of the dip solutions may vary within wide limits
depending, for example, dependant on the particular peptide
involved. However, it is generally preferred to formulate
relatively dilute solutions of the peptide.
The immersion time for the bulk material in the solution of the
peptide may vary depending on a number of factors. In general an
immersion time of from about 30 seconds to about 30 minutes,
preferably from 30 seconds to 15 minutes and in particular from 45
seconds to 5 minutes, has proven as valuable. The immersion of the
bulk material in the peptide solution may take place at room
temperature or at an elevated temperature; accordingly,
temperatures of, for example, from 15 to 30.degree. C. as well as
elevated temperatures of, for example, from 35 to 85.degree. C. are
possible.
A preferred embodiment of the invention comprises swelling the
hydrophobic organic bulk material in a water-miscible organic
solvent, for example, in a C.sub.1-C.sub.4-alcohol such as for
example ethanol or isopropanol or in THF, before treating it with
the solution of the peptide. The swelling may take place at ambient
temperature or preferably at an elevated temperature of, for
example from 35 to 90.degree. C. The swelling time is not critical;
usually a time period of from 30 seconds to 5 minutes, and
preferably from 45 seconds to 2 minutes is sufficient.
Following the deposition of the peptide the bulk material may be
worked up in an usual manner, for example by simple rinsing.
The hydrophilic surface coating (b) of the composite material
according to the process of the present invention may be finalized,
for example, chemically, by initiating the crosslinking of the
non-covalently bound peptide on the organic bulk material. To this
end the organic bulk material comprising the peptide on its surface
is treated with a suitable crosslinking agent, for example with
formaldehyde, preferably with an aqueous formaldehyde solution, or
with glutar aldehyde.
Preferably, the hydrophilic surface coating (b) of the composite
material according to the process of the present invention is
finalized by enzymatical crosslinking. For example, in case the
hydrophobic organic bulk material has attached to its surface a
peptide comprising both lysine and glutamine units, said peptide
may be crosslinked by the addition of a transglutaminase. The
enzyme transglutaminase initiates the formation of intrapeptide and
interpeptide isopeptide bonds between the lysine amino groups and
glutamine amido groups. The treatment of the bulk material with the
peptide attached to it in an aqueous solution comprising, for
example, bacterial transglutaminase may take place at ambient
temperature or preferably at a slightly elevated temperature of,
for example from 30 to 50.degree. C. The treatment time is not
critical; usually a time period of from 30 seconds to 10 minutes,
and preferably from 45 seconds to 5 minutes is sufficient.
In case the hydrophobic organic bulk material has attached to its
surface a peptide comprising lysine units only or glutamine units
only, enzymatical crosslinking using a transglutaminase is only
feasible in the presence of a further protein or protein
hydrolysate comprising the complementary amino acid units.
A further preferred embodiment of the invention therefore comprises
a process for the manufacture of a composite material comprising
the steps of (a) providing a hydrophobic organic bulk material, and
(b) applying a hydrophilic surface coating on said bulk material by
first (b1) attaching to the bulk material surface a peptide
comprising a hydrophobic moiety, which functions as a substrate for
the enzyme transglutaminase, preferably a compound of the
above-given formula (3), wherein the above-given meanings and
preferences apply for the variables contained therein; then (b2)
adding a protein or protein hydrolysate to the bulk material
surface which likewise functions as a substrate for the enzyme
transglutaminase; followed by (b3) treating with an enzyme, in
particular a transglutaminase.
For example, the peptide in step (b1) comprises one or more
glutamine units and the protein or protein hydrolysate in step (b2)
comprises one or more lysine units; or, in another embodiment of
the invention, the peptide in step (b1) comprises one or more
lysine units and the protein or protein hydrolysate in step (b2)
comprises one or more glutamine units.
It is believed that the transglutaminase treatment in the above
process fixes and/or crosslinks the protein or protein hydrolysate
on the peptide-modified surface of the hydrophobic bulk material.
Suitable proteins or protein hydrolysates in step (b2) above are,
for example, casein or casein hydrolysates, gelatine hydrolysates,
gluten hydrolysates or soy protein hydrolysates, in particular
casein hydrolysates. The treatment of the peptide-modified bulk
material according to step (b1) with the protein or protein
hydrolysate preferably takes place in an aqueous solution at
ambient temperature. It follows a treatment with the enzyme at
ambient temperature or preferably at a slightly elevated
temperature of, for example from 30 to 50.degree. C. The treatment
time is not critical; usually a time period of from 30 seconds to
10 minutes, and preferably from 45 seconds to 5 minutes is
sufficient.
The composite material obtained by the process of the invention
preferably is a biomedical device, e.g. an ophthalmic device,
preferably a contact lens including both hard and particularly soft
contact lenses, an intraocular lens or artificial cornea,
comprising a composite material as described above including all
the above given definitions and preferences. The composite
materials are further useful, for example, as wound healing
dressings, eye bandages, materials for the sustained release of an
active compound such as a drug delivery patch, moldings that can be
used in surgery, such as heart valves, vascular grafts, catheters,
artificial organs, encapsulated biologic implants, e.g. pancreatic
islets, materials for prostheses such as bone substitutes, or
moldings for diagnostics, membranes or biomedical instruments or
apparatus.
According to the process of the invention, biomedical articles, in
particular ophthalmic articles, are obtained that have a variety of
unexpected advantages over those of the prior art, which make those
articles very suitable for practical purposes, e.g. as contact lens
for extended wear. For example, they do have a high surface
wettability and lubricity. This can be demonstrated, for example,
by the finger tip test showing a very slippery article surface; or
by visual inspection; or by suitable contact angle measurements.
For example, sessile drop static contact angles of coated and
non-coated lenses are determined with a DSA 10 drop shape analysis
system from Kruss (Kruss GmbH, Hamburg, Germany). While uncoated
silicon hydrogel contact lenses in general have a water contact
angle of 90 to 100.degree. or above, a treatment according to the
process of the invention significantly reduces said value. Further
tools for assessing the superior quality of the surface coatings
obtainable according to the process of the invention are ATR-FTIR
measurements or the Sudan Black dye absorption test as described
below in the Examples section.
In addition, biomedical devices, e.g. ophthalmic devices such as
contact lenses, comprising a composite material obtained by the
process of the invention have a very pronounced biocompatibility
combined with good mechanical properties. In addition, there are
generally no adverse eye effects observed, while the adsorption of
proteins or lipids is low, also the salt deposit formation is lower
than with conventional contact lenses. Generally, there is low
fouling, low microbial adhesion and low bioerosion while good
mechanical properties can be for example found in a low friction
coefficient and low abrasion properties. Moreover, the dimensional
stability of the composite materials of the invention is excellent.
In addition, the attachment of a hydrophilic surface coating at a
given bulk material according to the invention does not affect its
visual transparency.
In summary, the ophthalmic devices obtained by the process
according to the invention, such as intraocular lenses and
artificial cornea or particularly contact lenses, provide a
combination of low spoilation with respect to cell debris,
cosmetics, tear components, lipids, proteins, salts, dust or dirt,
solvent vapors or chemicals, with a high comfort for the patient
wearing such opthalmic devices in view of the soft hydrogel surface
which for example provides a very good on-eye movement of the
ophthalmic device.
In the examples, if not indicated otherwise, amounts are amounts by
weight, temperatures are given in degrees Celsius. Wetting force on
the solid is measured as the solid is immersed in or withdrawn from
a liquid of known surface tension. The amino acid starting
materials as well as the amino acid units in the peptides are
always present in the naturally occurring L-form unless indicated
otherwise.
EXAMPLES
Example 1
Preparation of a Synthetic Polypeptide
(Tyr-Ala-Lys-Ala-Lys-Lys-Ala) of the SEQ ID NO 1
The above-mentioned peptide is synthesized at a polystyrene carrier
comprising 2-chlorotrityl anchors using standard methods of
Fmoc-solid phase peptide synthesis. Couplings are in general
performed in
o-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl-uroniumhexafluorophosphate
(TBTU)/1-hydroxy-1H-benzotriazol (HOBt), and the completeness of a
coupling is proven by the Kaiser test. Washing steps are performed
with N,N-dimethyl formamide (DMF).
Detailed Procedure:
(a) 6.25 g of a commercially available
Ala-OH-2-chlorotrityl-polystyrene carrier are suspended in 50 ml of
dichloromethane in a peptide synthesis reactor and kept for 30
minutes. The polymeric carrier is washed with DMF and afterwards
suspended again in some DMF. (b) Coupling of Fmoc-Lys(Boc)-OH: 4.69
g of Fmoc-Lys(Boc)-OH, 3.14 g of TBTU and 1.35 g of HOBt are
dissolved in 20 ml of DMF. 3.4 ml of N-ethyl-diisopropyl
amine(DIPEA) are added to this solution. The resulting mixture is
briefly stirred and is then added to the suspension obtained
according to step (a). The resulting mixture is maintained under
nitrogen for about one hour. Afterwards, a sample is taken and
checked by the Kaiser test. In case the Kaiser test is negative,
the reaction solution is extracted from the reactor, and the
polystyrene carrier is washed ten times with DMF. (c) Cleavage of
the Fmoc protective group: To the resulting polymeric carrier after
washing are added 30 ml of a DMF/piperidine mixture (80/20) and the
whole is kept for one hour while flushing with nitrogen. The
carrier is then again washed ten times with DMF and is afterwards
suspended in some DMF. (d) Coupling of Fmoc-Lys(Boc)-OH: In order
to add the second Lys(Boc) to the
Lys(Boc)-Ala-2-chlorotrityl-polystyrene prepared according to step
(c), steps (b) and (c) are repeated in an identical manner. (e)
Coupling of Fmoc-Ala-OH: 3.11 g of Fmoc-Ala-OH, 3.14 g of TBTU and
1.35 g of HOBt are dissolved in 20 ml DMF. 3.4 ml of DIPEA are
added to the resulting solution. The resulting mixture is briefly
stirred and is then added to the suspension obtained according to
step (d). The resulting mixture is kept under nitrogen for about
one hour. Afterwards, a sample is taken and checked by the Kaiser
test. In case the Kaiser test is negative, the reaction solution is
extracted from the reactor, and the polystyrene carrier is washed
ten times with DMF. It follows the cleavage of the Fmoc protective
group which is performed as described in step (c). (f) Coupling of
another Fmoc-Lys(Boc)-OH and Fmoc-Ala-OH: Both amino acids are
coupled to the peptide obtained according to step (e) in identical
manner as described in steps (d) and (e) above. (g) Coupling of
Fmoc-Tyr(tBu)-OH (tBu=tert.-butyl): 4.6 g of Fmoc-Tyr(tBu)-OH, 3.14
g of TBTU and 1.35 g of HOBt are dissolved in 20 ml of DMF and
afterwards 3.4 ml of N-ethyl-diisopropyl amine(DIPEA) added to this
solution. The resulting mixture is briefly stirred and is then
added to the suspension obtained according to step (a). The
resulting mixture is maintained under nitrogen for about one hour.
Afterwards, a sample is taken and checked by the Kaiser test. In
case the Kaiser test is negative, the reaction solution is
extracted from the reactor, and the polystyrene carrier is washed
with DMF. (h) Cleavage of the Fmoc protective group: To the
resulting polymeric carrier after washing are added 30 ml of a
DMF/piperidine mixture (80/20) and the mixture is kept for one hour
while flushing with nitrogen. The resin is then washed three times
with isopropanol and n-hexane and is afterwards dried in high
vacuum. Yield: 12 g of a polystyrene resin loaded with a synthetic
peptide of the SEQ ID NO:1 in which Tyr residue is protected with
t-Bu group and Lys residues are protected with Boc groups.
Example 2a
Coupling of a Hydrophobic Moiety to a Peptide Using Solid Phase
Chemistry
2.4 g of the polystyrene carrier loaded with a synthetic peptide of
the SEQ ID NO:1 in which Tyr residue is protected with t-Bu group
and Lys residues are protected with Boc groups obtained according
to Example 1 are suspended with dichloromethane in a peptide
reactor and kept for 30 minutes. The polystyrene carrier is then
washed with DMF and afterwards suspended again in some DMF. In a
separate jar 641 mg palmitinic acid, 786 mg TBTU and 338 mg HOBt
are dissolved in DMF. Following the addition of 850 .mu.l DIPEA and
thorough stirring this solution is added to the suspended
polystyrene carrier and the whole is maintained under nitrogen
flushing for about one hour. After the completeness of the
conversion has been confirmed by a Kaiser test, the polymeric
carrier is washed with DMF.
Example 2b
Coupling of a Hydrophobic Moiety to a Peptide Using Solid Phase
Chemistry
1 g of the polystyrene carrier loaded with a synthetic peptide of
the SEQ ID NO:1 in which Tyr residue is protected with t-Bu group
and Lys residues are protected with Boc groups obtained according
to Example 1 are suspended with dichloromethane in a peptide
reactor and kept for 30 minutes. The polystyrene carrier is then
washed with DMF and afterwards suspended again in some DMF. In a
separate jar 148 mg caprinic acid, 271 mg TBTU and 116 mg HOBt are
dissolved in DMF. Following the addition of 293 .mu.l DIPEA and
thorough stirring this solution is added to the suspended
polystyrene carrier and the whole is flushed with nitrogen for
about two hours. After the completeness of the conversion has been
confirmed by a Kaiser test, the reaction mixture is filtrated and
the polymeric carrier is washed with DMF.
Example 2c
Coupling of a Hydrophobic Moiety to a Peptide Using Solid Phase
Chemistry
910 mg of the polystyrene carrier loaded with a synthetic peptide
of the SEQ ID NO:1 in which Tyr residue is protected with t-Bu
group and Lys residues are protected with Boc groups obtained
according to Example 1 are suspended with dichloromethane in a
peptide reactor and kept for 30 minutes. The polystyrene carrier is
then washed with DMF and afterwards suspended again in some DMF. In
a separate jar 105 mg phenyl acetic acid, 239 mg TBTU and 103 mg
HOBt are dissolved in DMF. Following the addition of 261 .mu.l
DIPEA and thorough stirring this solution is added to the suspended
polystyrene carrier and the whole is flushed with nitrogen for
about one hour. After the completeness of the conversion has been
confirmed by a Kaiser test, the polymeric carrier is washed.
Example 2d
Coupling of a Hydrophobic Moiety to a Peptide Using Solid Phase
Chemistry
910 mg of the polystyrene carrier loaded with a synthetic peptide
of the SEQ ID NO:1 in which Tyr residue is protected with t-Bu
group and Lys residues are protected with Boc groups obtained
according to Example 1 are suspended with dichloromethane in a
peptide reactor and kept for 30 minutes. The polystyrene carrier is
then washed with DMF and afterwards suspended again in some DMF. In
a separate jar 141 mg naphthyl acetic acid, 239 mg TBTU and 103 mg
HOBt are dissolved in DMF. Following the addition of 261 .mu.l
DIPEA and thorough stirring this solution is added to the suspended
polystyrene carrier and the whole is flushed with nitrogen for
about one hour. After the completeness of the conversion has been
confirmed by a Kaiser test, the polymeric carrier is washed.
Example 3a
Cleavage of a the Fully Protected Peptide from the Polymeric
Carrier
From the polymeric carrier obtained according to Example 2a, the
fully protected peptide is separated. To this end 20 ml of a
solution comprising 1% by weight of trifluoroacetic acid in
dichloromethane are added to the polymeric carrier material and the
whole is shaken for about 2 minutes. The shaking process with the
trifluoroacetic acid/dichloromethane solution is repeated seven
times. Afterwards, the polymeric carrier is washed three times with
dichloromethane and methanol. The combined cleavage and washing
solutions are added to a solution of 10% by weight of pyridine in
methanol, and the whole is then concentrated in vacuum to a volume
corresponding to about 5% of the original volume. To the resulting
solution are added about 80 ml pure water and the resulting white
precipitate is filtrated. Following the repeated washing with cold
water, cold NaHCO.sub.3-solution, again cold water, cold 0.05M
KHSO.sub.4-solution and finally once again with water the
precipitate obtained is dried with P.sub.2O.sub.5 in vacuum
overnight. Yield 1.56 g of raw protected peptide.
Example 3b
Cleavage of the Deprotected Peptide from the Polymeric Carrier
The peptides as prepared according to Examples 2b, 2c and 2d are
separated from the polymeric carrier while removing the protective
groups of the side chains at the same time. To this end each 20 ml
of a solution comprising 2.5% by weight of water, 2.5% by weight of
tri-isopropyl silan and 95% by weight of trifluoroacetic acid are
added to the polymeric carrier comprising the respective protected
peptide and the whole mixture is then kept for about one hour. The
polymeric carrier is then filtrated off and is afterwards washed
twice with trifluoroacetic acid. Following the combination of the
filtrate and the washing solutions the solvent is removed in
vacuum. Crystallization of the resulting oily residue is initiated
by a treatment in diethyl ether. Yield (raw unprotected peptide, in
each case beige-colored crystals): caprinoyl-peptide in which the
peptide has the SEQ ID NO 1: 432 mg; phenac-peptide in which the
peptide has the SEQ ID NO 1: 370 mg; naphtac-peptide in which the
peptide has the SEQ ID NO 1: 395 mg.
Example 4
Coupling of palmitoyl-peptide in which the peptide has the SEQ ID
NO 1 in which Tyr residues are protected with t-Bu group and Lys
residues are protected with Boc groups to taurin
(2-sulfoethyl-amine)
780 mg of the peptide obtained according to Example 3a are
dissolved in 40 ml DMF. 160 mg TBTU, 68 mg HOBt, 340 .mu.l
N-ethyl-diisopropylamin and 125 mg taurin are added to this
solution and the reaction mixture is stirred overnight in a
nitrogen atmosphere; thereby the initial suspension turns into a
clear solution. Finally, the solvent is removed in vacuum, and the
solid residue is crystallized and washed with diethyl ether (yield:
1.4 g of a light brown solid).
The raw product, peptide in which the peptide has the SEQ ID NO in
which Tyr residue is protected with t-Bu group and Lys residues are
protected with Boc groups product is dissolved in 80 ml of a
solution comprising 25% by weight of trifluoroacetic acid and 75 by
weight of dichloromethane, and the whole mixture is stirred for
about 90 minutes at room temperature. The solvent is then removed
in vacuum and the remaining oily product is crystallized and washed
with diethyl ether.
Example 5
The raw products as obtained in Examples 3b and 4 are purified by
preparative HPLC(HPLC from Varian, reversed phase column with
water/acetonitrile gradient and trifluoroacetic acid as
modifier.
Yields and characterization:
(i) caprinoyl-peptide in which the peptide has the SEQ ID NO 1: 321
mg, ESI-MS: 955,6 [M+Na].sup.+
##STR00005## (ii) phenac-peptide in which the peptide has the SEQ
ID NO 1: 276 mg, ESI-MS: 919,5 [M+Na].sup.+
##STR00006## (iii) naphtac-peptide in which the peptide has the SEQ
ID NO 1: 260 mg, ESI-MS: 969,5 [M+Na].sup.+
##STR00007##
(iv) palmitoyl-peptide taurin in which the peptide has the SEQ ID
NO 1: 406 mg, ESI-MS: 1146,7 [M+Na].sup.+
##STR00008##
Example 6
Preparation of a Soft Silicon Hydrogel Contact Lens Having Attached
to its Surface a Peptide which is Enzymatically Crosslinked
A hydrophobic silicon hydrogel contact lens (lotrafilcon A,
copolymerization product of a mixed polysiloxane/perfluoroalkyl
polyether crosslinker, TRIS and DMA) is first incubated in an
aqueous solution comprising 10 mmol of the purified peptide of
Example 4 (palmitoyl-peptide-taurin in which the peptide has the
SEQ ID NO 1) and is then transferred to an aqueous solution
comprising 1% by weight of a casein hydrolysate (Vitalarmor).
Enzymatical crosslinking is initiated by the addition of 2 U/ml of
bacterial transglutaminase at 40.degree. C. The coated lens is then
washed with PBS buffer and autoclaved for 20 minutes at 121.degree.
C.
Example 7
Preparation of a Soft Silicon Hydrogel Contact Lens Having Attached
to its Surface a Peptide which is Chemically Crosslinked
(i) Preparation of the Silicon Hydrogel Contact Lens
(ia) Preparation of PDMS Crosslinker I
In a 4-L beaker, 24.13 g of Na.sub.2CO.sub.3, 80 g of NaCl and 1.52
kg of deionized water are mixed to dissolve. In a separate 4-L
beaker, 700 g of bis-3-aminopropyl-polydimethylsiloxane (Shin-Etsu,
MW ca. 11500) are dissolved in 1000 g of hexane. A 4-L reactor is
equipped with overhead stirring with turbine agitator and a 250-mL
addition funnel with micro-flow controller. The two solutions are
then charged to the reactor, and mixed for 15 minutes with heavy
agitation to produce an emulsion. 14.5 g of acryloyl chloride are
dissolved in 100 mL of hexane and charged to the addition funnel.
The acryloyl chloride solution is added dropwise to the emulsion
under heavy agitation over one hour. The emulsion is stirred for 30
minutes on completion of the addition and then agitation is stopped
and the phases are allowed to separate overnight. The aqueous phase
is decanted and the organic phase is washed twice with a mixture of
2.0 kg of 2.5% NaCl dissolved in water. The organic phase is then
dried over magnesium sulfate, filtered to 1.0 .mu.m exclusion, and
concentrated on a rotary evaporator. The resulting oil is further
purified by high-vacuum drying to constant weight. Analysis of the
resulting product by titration reveals 0.175 mEq/g of C.dbd.C
double bonds.
(ib) Preparation of PDMS Crosslinker II
In a 4-L beaker, 61.73 g of Na.sub.2CO.sub.3, 80 g of NaCl and 1.52
kg of deionized water are mixed to dissolve. In a separate 4-L
beaker, 700 g of bis-3-aminopropyl-polydimethylsiloaxane
(Shin-Etsu, MW ca. 4500) are dissolved in 1000 g of hexane. A 4-L
reactor is equipped with overhead stirring with turbine agitator
and a 250-mL addition funnel with micro-flow controller. The two
solutions are then charged to the reactor, and mixed for 15 minutes
with heavy agitation to produce an emulsion. 36.6 g of acryloyl
chloride is dissolved in 100 mL of hexane and charged to the
addition funnel. The acryloyl chloride solution is added dropwise
to the emulsion under heavy agitation over one hour. The emulsion
is stirred for 30 minutes on completion of the addition and then
agitation is stopped and the phases are allowed to separate
overnight. The aqueous phase is decanted and the organic phase is
washed twice with a mixture of 2.0 kg of 2.5% NaCl dissolved in
water. The organic phase is then dried over magnesium sulfate,
filtered to 1.0 .mu.m exclusion, and concentrated on a rotary
evaporator. The resulting oil is further purified by high-vacuum
drying to constant weight. Analysis of the resulting product by
titration reveals 0.435 mEq/g of C.dbd.C double bonds.
(ic) Preparation of the Crosslinkable Copolymer
A 2-L jacketed reactor is equipped with a heating/chilling loop,
reflux condenser, N.sub.2-inlet/vacuum adapter, feeding tube
adapter and overhead mechanical stirring. A solution is generated
by dissolving 90.00 g of PDMS crosslinker I according to (ia) and
30.00 g of PDMS crosslinker II according to (ib) in 480 g of
1-propanol. This solution is charged to the reactor and cooled to
8.degree. C. The solution is degassed by evacuating to less than 15
mBar, holding at vacuum for 15 minutes, and then re-pressurizing
with dry nitrogen. This degas procedure is repeated for a total of
3 times. The reactor is held under a blanket of dry nitrogen.
In a separate flask, a monomer solution is prepared by mixing 1.50
g of cysteamine hydrochloride, 0.3 g of AlBN, 55.275 g of DMA,
18.43 g of HEA and 364.5 g of 1-propanol. This solution is filtered
with a Whatman 540 filter paper, and then added to the reactor
through a degas unit and HPLC pump with a flow rate of 3.0
mL/minute. The reaction temperature is then elevated to 68.degree.
C. with a heating ramp about one hour.
In a second flask, a feeding solution is prepared by mixing 4.5 g
of cysteamine hydrochloride and 395.5 g of 1-propanol and then
filtering with Whatman 540 filter paper. When the reactor
temperature reaches 68.degree. C., this solution is slowly dosed
into the reactor through the degasser/HPLC pump over 3 hours. The
reaction is then continued at 68.degree. C. for an additional 3
hours, on which heating has discontinued and the reactor is allowed
to cool to room temperature.
The reaction mixture is transferred to a flask and stripped solvent
at 40.degree. C. under vacuum on a rotary evaporator until 1000 g
of sample remained. The solution is then slowly mixed with 2000 g
of deionized water with rapid agitation. Additional solvent is
further removed until about 2000 g of sample remain. During this
stripping process, the solution gradually becomes an emulsion. The
resulting material is purified by ultrafiltration over a 10 kD
molecular weight cut-off membrane until the permeate conductance is
below 2.5 .mu.S/cm. This emulsion is then charged to a 2-L reactor
equipped with overhead stirring, refrigeration loop, thermometer,
and the pH meter and dispensing tip of a Metrohm Model 718 STAT
Titrino. The reaction mixture is then cooled to 1.degree. C. 7.99 g
of NaHCO.sub.3 are charged to the emulsion and stirred to dissolve.
The Titrino is set to maintain pH at 9.5 by intermittent addition
of 15% sodium hydroxide solution. 11.59 mL of acryloyl chloride are
then added over one hour using a syringe pump. The emulsion is
stirred for another hour, then the Titrino is set to neutralize the
reaction mixture by addition of a 15% solution of hydrochloric
acid. The product is purified by ultrafiltration again with 10 kD
molecular weight cut-off membrane until the permeate conductance is
below 2.5 .mu.S/cm. The final macromonomer is isolated by
lypophilization.
(id) Preparation of Contact Lenses
18.83 g of the polymer obtained according to step (ic) are
dissolved in approximately 200 mL of 1-propanol, concentrated to
ca. 70 g total solution weight, and filtered to 0.45 .mu.m
exclusion. 67.94 g of solution at 26.53% solids are recovered.
4.503 g of a 1% solution of
2-hydroxy-4'-hydroxyethyl-2-methylpropiophenone
(IRGACURE.RTM.-2959, Ciba Specialty Chemicals) are added, and then
the solution is concentrated to a final weight of 25.74 g (65.0%
solids).
200 mg of the formulation are dosed into poly(propylene) contact
lens molds and the molds are closed. The molds are then irradiated
for 15 s with an ultraviolet light source having an intensity of
2.18 mW/cm.sup.2. The molds are then opened, and the mold halves
which have a lens attached are soaked in a mixture of 80%
isopropanol, 20% water (v/v) overnight. The lenses are rinsed off
the molds with this solvent mixture, then rinsed twice for 2 hrs.
each in fresh aliquots of isopropanol/water mixture. The lenses are
drained and then hydrated by immersion in deionized water. They are
then rinsed three times for 2 h in pure water (3.0 mL/lens).
(ie) Preparation of the Surface Coating
The hydrophobic silicon hydrogel contact lens obtained according to
(id) above is incubated in isopropanol for 1 minute at 75.degree.
C. and is then transferred into a phosphate buffered saline
solution comprising 10 mmol of the purified peptide of Example 4
(palmitoyl-Tyr-Ala-Lys-Ala-Lys-Lys-Ala-taurin) and treated for
about 1 minute at 80.degree. C. The contact lens is then
transferred into buffered saline and 100 .mu.l of a 2% by weight
formaldehyde solution are added. The contact lens is afterwards
autoclaved for 30 minutes at 121.degree. C. The attachment of the
peptide of Example 4 can be monitored by nitration reaction of the
thyrosine moiety in the peptide chain with tetranitro-methane
leading to a yellow staining. The hydrophilic surface coating is
investigated by visual wettability and hydrophilicity testing as
well as contact angle measurements and the Sudan Black staining
test.
(if) Water Contact Angle Measurement
The measurement is performed by the sessile drop method with a DSA
10 drop shape analysis system from Kruss GmbH, Germany with pure
water (Fluka, surface tension 72.5 mN/M at 20.degree. C.). For
measurement purposes a contact lens is taken off the storage
solution with tweezers and excess storage solution is removed by
gentle shaking. The contact lens is placed on the male part of a
contact lens mold and gently blotted with a dry and clean cloth. A
water droplet (about 1 .mu.l) is then dosed on the lens apex, and
the change of the contact angle over time of this water droplet
(WCA(t), circle fitting mode) is monitored; WCA is calculated by
extrapolation of the graph WCA(t) to t=0.
(ig) Sudan Black Dye Absorption Test
A 0.5% (w/w) Sudan Black dye solution is prepared by dissolving 0.5
g of Sudan Black B (Aldrich) over night in 100 g of vitamin E oil
under stirring. For measurement purposes, the surface-treated lens
is first of all autoclaved (30 min, 121.degree. C.) in 2 ml of an
phosphate buffered saline (pH 7.2) in a glass vial. The contact
lens is then removed from the solution with tweezers and gently
shaken so that most of the surface water is removed. The lens is
then placed in the above prepared Sudan Black dye solution for 5
min. Thereafter the lens is removed from the dye-bath, and the
excess dye solution is rinsed off with warm water. The lens is
air-dried and assessed according to its degree of staining. 2=no or
almost no staining 1=slight staining 0=considerable staining (ih)
The Values Obtained with Contact Lenses as Coated According to (ie)
and with the Corresponding Uncoated Contact Lenses (Control) are
Summarized in Table I
TABLE-US-00001 Example WCA [.degree.] Sudan Black ie 58 2 (Control)
109 0
SEQUENCE LISTINGS
1
117PRTArtificial Sequencesynthetic 1Tyr Ala Lys Ala Lys Lys Ala1
5
* * * * *